Project summary: Signaling through cyclic AMP (cAMP) and its effector molecules, such as cAMP-dependent protein kinase (PKA) and exchange proteins activated by cAMP (Epac), regulates a variety of cellular functions including cell growth, proliferation, metabolism, survival and mobility, as well as insulin secretion in the case of pancreatic ? cells. The overall goal of our research is to elucidate the molecular mechanisms and functional roles of spatiotemporal regulation in achieving high specificity in cAMP signaling. Aberrations in the cAMP signaling pathway have implications for clinical conditions such as obesity and type 2 diabetes mellitus, particularly in the context of ?-cell functions. A mechanistic understanding of cAMP signaling specificity is crucial to developing therapeutic strategies for these clinical conditions. The concept of spatial compartmentalization of cAMP effects was proposed 20 years ago, but only in recent years have innovative approaches to studying cAMP signaling in the cellular context become available to provide direct mechanistic evidence. However, despite these recent advances, there are still large gaps in our understanding about the mechanisms underlying the spatiotemporal regulation of cAMP and its effectors. Furthermore, little is known about how the signaling information encoded in the spatiotemporal patterns of activities is translated into specific functional responses. In our preliminary studies, we have developed new molecular tools to monitor and perturb cAMP/PKA activities in living cells with further enhanced spatiotemporal resolution and precision. Furthermore, building on our recent discovery of a Ca2+-cAMP-PKA oscillatory circuit in MIN6 ? cells, we showed that A-Kinase Anchoring Protein 79/150 (AKAP79/150) assembles a PKA-containing signaling complex in these cells and influences the activity dynamics of the Ca2+-cAMP-PKA circuit. In the current proposal, utilizing our new molecular tools and by combining computational modeling and experimental approaches, we will test our hypothesis that the Ca2+-cAMP-PKA oscillatory circuit, further regulated spatially and temporally by AKAPs, allows PKA to achieve high signaling specificity and diversity through frequency modulation. The specific aims are: 1) developing novel molecular tools to interrogate the spatiotemporal regulation of cAMP/PKA signaling in living cells; 2) elucidating the spatial compartmentalization and frequency control of the Ca2+-cAMP-PKA oscillatory circuit.